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From 2010.igem.org

Contents 1 Sterile Agar Plate Preparation and Seed Placement 2 Materials 3 Methods 4 Notes

Sterile Agar Plate Preparation and Seed Placement

Seed sterilization link

Agar prep link

Materials

1. sterile seed in deluted agar
2. sterile agar media
3. sterile hood environment
4. P1000 pipeter
5. blue pipeter tips
6. plates
7. parafilm tape
8. 70% ethanol solution in spray bottle
9. grid template for desired seed alignment (to be placed under transparent media and referenced during seed placement)

Methods

Pouring Agar

1. sterilize items one by one with 70% ethanol spray, placing each item into sterile hood after being sprayed.
2. once all are in hood, remove aluminum foil wrap from agar flask.
3. remove lid off first plate and keeping the lid as a shield between you and the open plate, pour agar into plate. Fill to halfway point, carefully avoiding air bubbles in media. Place lid right-side-up on hood surface, then set poured plate on top. Allow to cool before replacing lid as to avoid condensation.
4. repeat for remaining plates.

Seed Placement

1. draw horizontal line across plate with a center point roughly one inch from the perimeter of plate
2. place reference grid (link to pic or file) under plate, matching horizontal lines.
3. set pipeter to small volume(?) so that when fully released the seed mixture level remains within the lower angled section of the blue tip.
4. after pulling a small amount of seed (#3) into blue tip, remove tip.
5. remove lid off first plate and keeping the lid as a shield between you and the open plate.
6. to place seed, reference the grid below the agar and gently touch surface of agar with tip, so that only one seed is placed onto the agar. (Too little seeds in tip will result in no seeds coming out of tip, and too much seed mixture will cause a group of seeds to come out of tip onto agar). If seeds do not come easily from tip, pressing one's finger into top of tube may work to create pressure and dislodge seed.
7. place seeds at desired density.
8. replace lid and move to next plate. Repeat steps.
9. when all plates are finished, seal with parafilm tape.
10. plates can be placed horizontal or vertical in growth room

Notes

Keep head out of hood. Contamination from hair, mouth, nose, hands, etc. will infect culture plate with mold spores.